Treatment of proteins



Patented July 26, 1949 TREATMENT Havard K'eihfllamendon Hills,

ling Ingmh mr tion. of Illinois andJ'oseph Ster- Ghicago, IlL, assignors to Armourandfiompany,

Chicago,- lllt, w corpora- Nor Drawing. Application October I0, 1947; Serial]: No. 779,228

8. Claims, (Cl. 252 -3llfi Thisginvention relateslto the treatment of. pro..-

and. morevv particularly to; a. process for hydrolyzing; proteinszandsfor preparinga proteinaceous foam stabilizer. The inventionalsov deals. withza neywhydrolysate, product. and the improved mam-stabilizer. This case constitutesa continuation-in-part of our co-pending application, Serial 565,5124e (abandoned)-, for Treatment of pro.-

Bnoteinaceous foam stabilizers for the extineuishinerot fires are known to have many advantages. These: materials; produce a foam of fine, bubblestnuctnre: whichis also stable. to relatively. intense heat. It is also known that by; incorporating salts of heavy-polyvalent metals in proteinaceous foam stabilizers, the bubble film of the foammay be further strengthened and the-resistance of the foam to fire further increased; Difltculties'; however; may be encountered in; in-- corporating the protein-aceousfoamstabilizer the desiredouantityofisalts of heavypolyvalent metals since these salts tend toform insoluble: complexes: inthe: protein hydrolysate, and: the solubility oi such: salts is subject to numerous limitations;

By the present process, a protein hydrolysate; adapted for use as=a foam stabilizer is formed-iwitln a minimunr quantity ofinorganic salts present.- im the. hydrolysate. This permits; the incorporat tion of larger. quantities of salts of heavy; poly.-

valent: metals: than. would otherwise be; possible. As proteim hydrolysateproduced in accordance: with'tha invention also be; substantially-free Qfr'minerakacids and the salts-.formedby neutraliezatiomof; these acids-which also permitathe. ina corporation of additional quantities; of heavy metisaitgirr the product. The enzyme hydrolysa-te maybeaproducedz from; blood proteins which have;

henetoiore not. been economically, and efi'eotively hydrolyzed with enzymes. In addition, salts. oftheiheavy-polyvalent meta-ls are used whichper.- mit a maximum. incorporation of the metallicmns.

The, invention contemplates. the formation. at. afoamh-stabilizing protein hydrolysate in which. maybe. incorporated. a maximum quantity. of the salts of heavy polyvalent metals, and, further contemplates the effective and efiicient enzyme hydrolysismf, blood proteins.

The. invention). is applicable. to. any suitable proteins, such. as. the histones, protamines, globins,.globulins, albumins, keratins, casein-,,m-ucin,. prolamines, and gluten. The invention, however, isnarticularly. applicable to blood proteins.

The proteins are hydrolyzed by enzyme by- 2?: drel ysis; since blood proteins are not hydrolyzed by norma-P enzyme hydrolysis,- these proteins in: accordance with: the invention aresubjected to a pretreatment in which theproteins are condi tionedfor-enzymehydrolysis: It-is beli'eved thatl' there are certain elements or-fact'ors present intheblood proteins which interferewith enzyme hydrolysis oftheseproteihs; These elements arereferred -to hereinas-anti--enzymes; and*'-'thepretreatment' is designed to destroy these: anti-enzymes:

In the case of 'keratins; it is: also:-- necessary to destroy antiienzymes-before the; hydrolysis can-be eifectively' carried out;

Inthecase of proteins other than blood pro ins and" the keratins it is possible, to hydrolyze the-proteins with enzymes withoutithepretreatimeat, but the alkali" pretreatmentof', the" inverttion" has" been" found to; condition the proteins: for enzyme hydrolysis: so as tomake, thehydrolysis, easier, more economical and" more: effective:

The; pretreatmentof the proteins may involve, heating the proteinsi'n an alkalineimedium by; which is. meant, anaqlieous solution of; an. alkaline earth, metal, or; alkali metal oxide. or. Pay? droxicl'e. Preferably an. alkali; metal hydroxide; such as sodium.hydroxitie,. isused; The, quantity. ofthe, alkali .metalihrdroxide preferably, inmthe range of.i3'% to.9%'. by weightwithrespeqt.tathe dry protein. 7.5%. has beenioundltoebe very sate isfactory...

The; alkali pretreatment: be, carried. on'efor a sufiicient length. of time, to... destroy the. anti: enzymes. Normall'y a. period ot time. inv the neighborhood of one-hour. has. been found tohe sufficient, butv a somewhat; longer period? ofi'time will usually permit morerapii action oi the em Zymes. when the, enzymehydrolysis isecarried aouti After. the proteins have been treated-with 1 aka-- lilto, conditiontthe. proteins for emzvme. hydrolysis, the, excess. alkali.

It. is. preferred that mineral. acids. not be used since. the, usev oi. thesev acids; tends. to. result in .aprecipitation. of. heavy, metal. salts ilnthe form-0f protein .:complexes. when the. heavy metal. salts. are. later. added. In: preparing. a foamestabilizing. agent, organic= acids are used rather thanmineral-lacidsineanyneutralizationeyen if theprotein isv onev which: may; bet-normally enzyme hydrolyzed.

The neutralized pretreated; protein-.. product is subjected to enzyme hydrolysis under conditions of temperature and hydrogen ion concentration favorable to the quantity of the enzyme used. Any suitable hydrolyzing proteolytic enzyme may be used. For example, pancreatin or papain is suitable, In the case of these enzymes, a temperature in the neighborhood of 40 C. and a pH of about 5 to 8 have been found tobe satisfactory.

The enzyme hydrolysis is carried on until a foam-form protein hydrolysate is formed. Preferably, the time of the enzyme hydrolysis is between 12 and 24 hours, but this time mayvary considerably with the quantity of the'enzyme used. I

The enzyme hydrolyzed protein contains insoluble constituents and these materials are separated from the mixture by filtration or other 7 suitable process.

To the protein hydrolysate may be added a salt of a heavy polyvalent metal, such as iron, chromium, nickel, aluminum, calcium, barium, copper, lead or arsenic. The metal is preferably one which exists in more than one state of valence, and in this case is preferably added in a form in which the metal is in a lower state of valence. For example, ferrous sulfate, ferrouschloride or other ferrous salts may be used. Preferably, the sulfate is used, as this has been found tobe more readilyincorporated than other ferrous salts, such as the chloride and acetate.

If'a very large quantity of the salt of the heavy'polyvalent metal is to be incorporated in the protein hydrolysate, the mixture maybe adjusted to a pH of between 3 and 5.5, preferably between 4.5 and 4.8, and the precipitate formed separated by filtration. This operation is carried out after the enzyme hydrolysis and before the addition'of the heavy metal salt. The precipitate apparently consists of high molecular weight protein degradation products fwhich interfere.

with the incorporation of large quantities of the heavy metal salt. Without this action, between 4% and 8% of a heavy metal salt, such asferrous metal. sulfate, may be readily incorporated in a blood protein sulfate prepared in accordance with the invention. If, however, the heavy molecular weight protein degradation products are removed, as high'as 12% by 'weight of the ferrous sulfate may be incorporated in the mixture.

After the enzyme hydrolysis, the mixture may be heated to inactivate the enzyme. The mixture may also'be chemically treated in any conventional manner to inactivate the enzymes.

.The' solids content of the proteinaceousfoamstabilizing agent s preferably in the neighborhood of 30%. When the heavy molecular weight protein degradation products'have not been .removed, the viscosity of the solution will be relatively high as compared to the fractionated productfromwhich the higher molecular weight degradation'products'have been removed. In either case, the viscosity ofthe solution may be adjusted by changing the concentration thereof. However, in the case of the mixture containing the high molecular weightproteindegradation products, smallchanges in concentration produce a muchjgreaterchangein viscosity than in the,

case of the fractionated product. p

v The enzyme hydrolyzed protein,'wheth'er blood protein or other 'protein,'has been found to be particularly suitable for use as a foam-stabilizingagent since the enzyme hydrolysis does not destroy as many amino acids as does alkali or acid hydrolysis and the nitrogen content of the hydrolysate is higher.

' In the pretreatment of the proteins in an aling,

'4 kaline solution, we prefer to employ a temperature of from 90 to 100 C. or higher. By bringing the proteins in the alkaline medium to a temperature within this range and preferably to the boiling temperature, we find that the antienzymes are rendered ineffective, and in the ensuing enzyme hydrolysis, the hydrolysis is effectively carried out.

The following are vention:

specific examples of the in- E'xample I Liquid blood or dry blood mixed with approximately 7 times its own weight of water is mixed with approximately 7.5% of sodium hydroxide based on dry protein weight, and the mixture heated for a period of 1 to 5 hours. After heatthe solution is neutralized to a pH of about 8 by the addition of acetic acid. To the solution is then added approximately 3% of pancreatin, the proportion being based on the dry protein weight; The mixture is permitted to stand for a period of 12 to 24 hours at a temperature of 40 C. and a pH of 8 to carry out the enzyme hydrolysis. V

The enzyme hydrolysate is adjusted in pH to approximately 7.5 and heated to boiling to inactivate the enzyme. solution is adjusted by evaporation'or the addition of water to a solids content of approximately 30%. The solution is filtered to remove insoluble constituents and between 4% V rous sulfate is then added thereto. 'The final pH of the solution may be in the-neighborhood of 6.3+6.'7. a

Ezmmple II The enzyme hydrolysateprepared by the above.

method may, after heating to stop enzymic action, be acidified with sulfuric acid to a :pH of, 4.5 and heated to boiling. A heavy gummyprecipitate separates out, and this precipitate is removed by decantation, filtration, or the like. The mixture is then neutralized to 7.5, using lime or calcium hydroxide to remove the sulfuric acid as calcium sulfate. The solution is again boiled and filtered free of a small precipitate. The concentration is then adjusted by evaporation or dilution until the solids content is approximately 30% and between 4% and 12% ferrous sulfate added to the solution. i In each'of the above examples sodium carbonate and tri-sodium phosphate may be substituted for-the sodium hydroxide, and satisfactory re-- sults will be obtained. We prefer, howeveigtosirable foam-stabilizing product.-

Although the invention has beenillustrated in connection with certain specific embodiments, it will be apparent that, modifications'and changes may be made without departing from the spirit and scope of the invention.

We claim:

1. A process of preparing. a proteinaceous foam-stabilizing composition, comprising heating ablood protein in an aqueous solution, of sodium hydroxide to. a temperature ofat least C. to destroy anti-enzymes, subjecting the mixture to.

hydrolysis with a hydrolyzingproteolytic enzyme The specific gravity of the and 8 of fer-' by weight of under conditions of temperature and hydrogen ion concentration favorable to the enzyme action, and adding to the remaining solution from 4 to 8% of a water-soluble salt of a heavy polyvalent metal.

2. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to a temperature of about 90-100 C. a blood protein in an aqueous solution of sodium hydroxide to destroy anti-enzymes, subjecting the mixture to hydrolysis with a hydrolyzing proteolytic enzyme under conditions of temperature and hydrogen ion concentration favorable to the enzyme action, adjusting the pH to 3-5.5 to form a precipitate, removing the insoluble material, and adding to the remaining solution from 4 to 12% of a water-soluble salt of a heavy polyvalent metal.

3. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to a temperature of about 90100 C. a blood pro tein in an aqueous solution of sodium hydroxide to destroy anti-enzymes, changing the pH of the mixture to that favorable to the action of a selected enzyme by the addition of an organic acid, subjecting the mixture to hydrolysis with said selected proteolytic enzyme under temperature conditions favorable to the enzyme action, adjusting the pH to 3-5.5 to form an insoluble precipitate, separating the insoluble material, and adding to the remaining solution from 4 to 12% of a watersoluble salt of a heavy polyvalent metal.

4. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to about boiling temperature a blood protein in an aqueous solution of sodium hydroxide to destroy anti-enzymes, changing the pH of the mixture to that favorable to the action of a selected enzyme, subjecting the mixture to hydrolysis with said selected proteolytic enzyme under temperature conditions favorable to the enzyme action, adjusting the pH to 3-5.5 to form a precipitate, removing the precipitate, and adding to the solution from 4 to 12% of a water-soluble salt of a heavy polyvalent metal.

5. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to about a boiling temperature blood in an aqueous solution of sodium hydroxide to destroy anti-enzymes, adding an organic acid and a selected proteolytic enzyme, subjecting the mixture to hydrolysis with said selected proteolytic enzyme and under temperature conditions favorable to the enzyme action to produce a foamforming protein hydrolysate, removing the insoluble material, and adding to the hydrolysate from 4 to 8% of a Water-soluble salt of a heavy polyvalent metal.

6. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to about a boiling temperature a blood protein in an aqueous solution of sodium hydroxide to destroy anti-enzymes, subjecting the mixture to hydrolysis with a hydrolyzing proteolytic enzyme nder conditions of temperature and hydrogen ion concentration favorable to the enzyme action to produce a foam-forming protein hydrolysate, adjusting the pH to about the range of 4.5-4.8 to precipitate degradation products, removing the precipitate, and incorporating with the hydrolysate from 4 to 12% of a water-soluble salt of a heavy polyvalent metal.

7. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to about a boiling temperature blood in an aqueous solution of sodium hydroxide to destroy antienzymes, adjusting the pH through the use of an organic acid to that favorable to the action of a selected proteolytic enzyme, subjecting the mixture to hydrolysis with the selected proteolytic enzyme under temperature conditions favorable to the enzyme action to produce a foam-forming protein hydrolysate, adjusting the pH to about 4.5-4.8 to produce a precipitate of high molecular weight protein degradation products which interfere with the incorporation of large quantities of a heavy metal salt, removing the precipitate, and incorporating in the hydrolysate from 4 to 12% of a water-soluble salt of a heavy polyvalent metal.

8. A process of preparing a proteinaceous foam-stabilizing composition, comprising heating to about a boiling temperature a blood protein in an aqueous solution of sodium hydroxide to destroy anti-enzymes, subjecting the mixture to hydrolysis with a hydrolyzing proteolytic enzyme under conditions of temperature and hydrogen ion concentration favorable to the enzyme action to produce a, foam-forming protein hydrolysate, adjusting the pH of the solution to between 3 and 5.5 to produce a precipitate of high molecular Weight protein degradation products which interfere with the incorporation of a water-soluble salt of a heavy polyvalent metal, removing the precipitate, and incorporating in the hydrolysate from 4 to 12% of a, water-soluble salt of a heavy polyvalent metal.

HAVARD L. KEIL. JOSEPH STERLING INGRAHAM.

REFERENCES CITED The following referenlces are of record in the file of this patent:

UNITED TATES PA'I'EN'IW'S Number Name Date 1,754,825 Heuser Apr. 15, 1930 2,151,398 Weissenborn Mar. 21, 1939 2,158,499 Grassmann et a1. May 16, 1939 2,212,470 Friedrich Aug. 20, 1940 2,324,951 Ratzer July 20, 1943 2,381,407 Levinson et a1 Aug. 7, 1945 2,405,438 Levin Aug. 6, 1946 2,431,256 Keil et a1. Nov. 18, 1947 

